Investigation of Novel Nanoparticles of Gallium Ferricyanide and Gallium Lawsonate As Potential Anticancer Agents, and Nanoparti

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Investigation of Novel Nanoparticles of Gallium Ferricyanide

and Gallium Lawsonate as Potential Anticancer Agents, and

Nanoparticles of Novel Bismuth Tetrathiotungstate as

Promising CT Contrast Agent

A Thesis submitted

to Kent State University

In partial fulfillment of the requirements for the

degree of Master of Science

Liu Yang

August 2014

Thesis written by

Liu Yang

B.S. Kent State University, 2013

M.S. Kent State University, 2014

Approved by

______, Advisor, Committee member

Dr. Songping Huang

______, Committee member

Dr. Scott Bunge

______, Committee member

Dr. Mietek Jaroniec

Accepted by

______, Chair, Department of Chemistry

Dr. Michael Tubergen

______, Dean, College of Arts and Sciences

Dr. James L. Blank

Table of Contents

List of Figures..…………………………………………………………………...... vii

Acknowledgements ……………………………………………………………….….xi

Chapter 1: Summary, Materials and Methods …..……………………………………1

1.1 Materials ………………………………………………………………….3

1.1.1 carboxymethyl reduced polysaccharide (CMRD) preparation….3

1.2 Methods …………………………………………………………………4

1.2.1 Atomic absorption spectroscopy (AA) …………………………4

1.2.2 Acid base treating method ……………………………………...4

1.2.3 Cell viability study ……………………………………………...5

i) MTT assay…………………………………………………..5

ii) Trypan blue assay ………………………………………….6

1.2.4 Dialysis …………………………………………………………6

1.2.5 Elementary analysis …………………………………………….7

1.2.6 Lyophilization …………………………………………………..7

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1.2.7 Kinetic study ……………………………………………………7

1.2.8 Thermal gravimetric analysis (TGA) ………………………..…8

1.2.9 Selectivity study ……………………………………….……..…8

1.2.10 Nanoparticle-surface conjugation of fluorescence dye

molecules ...….………………………………………………………..9

1.2.11 X-ray attenuation measurements and CT phantom imaging…..9

1.2.12 Fourier transform infrared spectroscopy (FTIR)……………..10

1.2.13 Transmission electron microscopy (TEM) and energy

dispersive X-ray (EDX)……………………………………………...10

Chapter 2: Novel Gallium Ferricyanide Nanoparticles as Potential Anticancer

Drug …………………………………………………………………………………11

2.1 Abstract ………………………………………………………………….11

2.2 Introduction ……………………………………………………………...11

2.3 Synthesis and characterization of bulk gallium ferricyanide compound...19

2.3.1 Method A………………………………………………………19

2.3.2 Method B………………………………………………………21

2.3.3 Results and discussion of Ga[Fe(CN)6] bulk compound………25

2.4 Synthesis and characterization of gallium ferricyanide nanoparticles…..27

2.4.1 Synthesis of gallium ferricyanide nanoparticles……………….27

2.4.2 Results and discussion ………………………………………...28

2.5 Conclusion……………………………………………………………….37

Chapter 3: Gallium Lawsonate Nanoparticles as Potential Anticancer Agent………38

3.1 Abstract …………………………………………………….……………38

3.2 Introduction ……………………………………………………………...38

3.3 Synthesis …………………………………………………….…………..43

3.4 Results and discussion ……………………………………….………….46

3.5 Conclusion ………………………………………………………………47

Chapter 4: Bismuth Tetrathiotungstate Nanoparticles as Potential Contrast Agent for

Computed Tomography……………………………………………………………...48

4.1 Abstract ………………………………………………………………….48

4.2 Introduction ……………………………………………………………...48

4.3 Synthesis ………………………………………………………………...51

4.4 Results and discussion …………………………………………………..51

4.5 Conclusion ……………………………………………………………....56

Chapter 5: References ……………………………………………………………….57

List of Figures

Figure 1. Iron uptake through transferrin receptor (TfR). Fe 2+ is oxidized to Fe 3+ and bounded to transferrin protein (Tf). Diferric Tf binds to the receptor and internalized with clathrin coating through receptor mediated endocytosis. In the endosome, at lower pH, Fe3+ is released from transferrin protein and reduced back to Fe2+. Fe2+ can cross the membrane through ion channel and become available for other cellular process………………………………………………………………………………17

Figure 2. Ga3+ uptake mimicking the Fe3+ uptake pathway: Ga3+ crosses plasma membrane with the help of transferrin protein and transferrin receptor. Afterwards in the acidic endosome, Ga3+ is released from transferrin and enters cytoplasm………18

Figure 3. (up) Junction of endothelial cells in normal and healthy blood vessels is 5-

10 nm. NP (>10 nm) would pass through. (Bottom) Junction of endothelial cells in abnormal blood vessels is usually few hundred nanometers, NP(<100 nm) would leak out from abnormal blood vessels and target tumor tissue. ………………………….19

Figure 4. Fourier transform infrared spectroscopy (FT-IR) of the Ga[Fe(CN)6] bulk compound synthesized by method A………………………………………………...20

Figure 5. TGA of Ga[Fe(CN)6] bulk compound synthesized by method A. Weight lost below 200 ºC showed on the first drop on graph was due to the water lost, The

vii

second drop showed on graph above 200 ºC may due to cyanide lost. 21% weight loss was due to the water………………………………………………………………….21

Figure 6. TGA of Ga[Fe(CN)6] bulk compound synthesized by method B. Weight lost below 200 ºC showed on the first drop in the graph was due to the water lost.

The second drop showed in the graph above 200 ºC may be due to cyanide lost.

21%weight loss was due to the water……………………………………………….23

Figure 7. FT-IR of Ga[Fe(CN)6] bulk compound synthesized by method B………24

Figure 8. The Fe(III) center donates electron through a d orbital to empty π*, an anti-bonding orbital of the CN ligand………………………………………………24

Figure 9. The formula used for calculating water molecules in crystal lattice. X represents the number of water molecules…………………………………………...25

Figure 10. FT-IR of gallium ferricyanide nanoparticles in comparison with gallium ferricyinde bulk compound and the CMRD polymer coating agent…………………29

Figure 11. Transmission electron microscopy (TEM) images of CMRD coated gallium ferricyanide nanoparticles with an average size of 25 nanometers…………29

Figure 12. Selectivity studies. Ga NPs undergo ion-exchange with 100 ppm of several M(II) ions for 24 hours showing that Fe(II) to be the most selective metal and

Mn(II)/Mg(II) the least selective metals……………………………………………..30

Figure 13. Kinetic study. (Top) Fe2+ removal by Ga NPs vs. time. (Bottom) Within first 20 min, the removal of Fe2+ fits the pseudo-first order reaction………………...31

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Figure 14. A: Preparation of fluorescence Ga NPs. B: Confocal microscopy images of T24 cells: (Top left) fluorescence image of cells incubated with dye-conjugated Ga

NPs for 3 hours; (Top right) bright field images of cells incubated with dye conjugated Ga NPs for 3 hours; (lower left) florescence images of the cells untreated with NPs; (lower right) bright field image of the cells untreated with NPs……….33

Figure 15. Confocal microscopy images of HT-29 cells: (Top left) fluorescence image of cells incubated with dye-conjugated Ga NPs for 3 hours; (Top right) bright field images of cells incubated with dye conjugated Ga NPs for 3 hours; (lower left) florescence images of the cells untreated with NPs; (lower right) bright field image of the cells untreated with NPs…………………………………………………………34

Figure 16. Cell viability curve based on the MTT assay. Effect of Ga NPs on viability of HT-29 cells after 24 hour-incubation in comparison with the cell viability using Ga(NO3)3………………………………………………………………………35

Figure 17. Bright field light microscopic images. Various concentrations of Ga NPs used for incubating HT-29 cells for 24 hours. A: 1.58mM; B: 2.3mM; C: 3.15mM; D:

3.94mM; E: 4.73mM; F: 5.51mM; G: 6.3mM; H: NP-free control cells……………36

Figure 18. Structures of the oral gallium maltolate (right) and maltol……………..42

Figure 19. Structure of the oral KP46………………………………………………42

Figure 20. Structure of lawsone……………………………………………………..43

Figure 21. Synthetic scheme and proposed structure of gallium lawsonate………..44

Figure 22. Cell viability studies of Ga-lawsonate NPs on HT-29 Cells (Top) and T24 cells (Bottom) by MTT method …………………………………………………….45

Figure 23. Cell viability studies of Potassium lawsonate on T24 Cells……………46

Figure 24. (TOP) Transmission electron microscopy (TEM) image of Bi2(WS4)3 NPs on the 5-nm scale. (Bottom) Energy dispersive X-ray spectrum of Bi2(WS4)3 NPs;

TEM analysis revealed that the nanoparticles are well-formed cubes and the size distribution appeared to be relatively wide, ranging from 2 to 10 nm……………….53

Figure 25: Fluorescence microscopic images of HeLa cells incubated with dye- labeled NPs for 3 hours: (right) Bright-field image, (left) confocal fluorescence microscopy image; Carboxyfluorescene dye was conjugated to the surfaces of the ethylenediamine-coated Bi2(WS4)3 NPs by the EDC-coupling reaction. Fluorescent signal in the cytoplasm of cells confirms the uptake of the NPs by the cells. Therefore these agents can serve as intracellular contrast agents……………………………….54

Figure 26. Histogram showing the viability of Hela cells in the presence of various amounts of Bi2(WS4)3 NPs after 24 hours of incubation as determined by the Trypan

Blue exclusion method. ……………………………………………………………...55

Figure 27. Phantom images of Bi2(WS4)3 NPs at different concentrations………….55

Figure 28: The CT values vs. Bi(III) concentrations………………………………..56

Acknowledgements

I specially would like to thank Dr. Songping Huang for his years of patience and advice. Without his knowledge and assistance, I probably could not solve problems I met and keep the projects going. And I learned a lot from doing research these years.

I would like to thank most senior lab member Vindya Perera for her help with

TEM and confocal microscopy. And she also helped me to learn dialysis and lyophilization methods.

I would like to thank Murthi S. Kandanapitiye who trained me with atomic absorption, kinetic studies, TGA, cell culture, trypan blue method and data analysis.

I would like to thank Karen from Summa hospital who trained me for MTT assay.

I would like to thank Dr. Min Gao from Liquid Crystal Institute for providing

TEM training.

Additionally, I would like to thank the Chemistry and Biochemistry Department

Secretaries Erin Michael and Pamela (Janie) Viers for their assistance.

Finally, I would like to thank NIH-NCI for financial support (1R21CA143408-

01A1) and NSF-CRIF for a grant (CHEM1048645) to update the departmental NMR facility. Moreover, thanks for Dr. Twig’s, Dr. Brasch’s, and Dr. Fraizer’s research lab for sharing their instruments.

Chapter 1: Summary, Materials and Methods

Summary of projects

Gallium is a main group element. After platinum, gallium was a second metal found has anticancer properties in early 1970s. Gallium is non-toxic and redox inactive. Because of similar ionic radii, electronegativity, ionization potential and electron affinity, gallium ions could be looked as a mimic of ferric ions. Thus, gallium ions are able to interference iron metabolism, especially intracellular process.

Ferric ion is a cofactor of ribonucleotide reductase of DNA replication, once ferric ion is replaced by gallium ion, ribonucleotide reductase would be inhibited. This would greatly affect cell division. Due to anticancer properties, gallium compounds are developed as anticancer agent to treat solid tumor. The first generation of gallium anticancer agent was gallium nitrate as an oral drug. However, there are some problems of gallium nitrate. Gallium nitrate is a metal salts which has low efficiency and bio-availability in cancer treatment. Patents needed to take large doses of gallium nitrate to produce desired results. On one hand, free gallium ions may form hydroxide in biological pH. And it could be cleared by kidney in few hours easily. Large dose of gallium nitrate may cause some damage to kidney. On the other hand, tumor cells start to build up drug resistance by either shutting down or turning off transferrin receptor activity or excreting gallium ions through ion pump. To solve these problems,

scientists are investigating gallium complexes as small molecules or nanoparticles for drug delivery.

We developed gallium ferricyanide nanoparticles and gallium lawsonate nanoparticles as potential anticancer agents to treat solid tumor. Gallium ferricyanide is an analog of Prussian blue which found in 1706 by an artists’ color maker, Heinrich

Diesbach. It was used as a pigment in painting for a long time. It was known that

Prussian blue is non-toxic. It could use to treat thallium poisoning and as cesium removal.

Gallium ferricyanide nanoparticle was investigated regarding its physical properties and anticancer properties to cancer cells.

Beside anticancer agents, a potential CT contrast agent was developed.

Computed tomography (CT) is a technique for diagnosing diseases using X-ray radiations.

CT contrast agents are usually used as “dyes” to highlight specific areas. So CT contrast agents have high X-ray attenuation coefficient which contributes to visualization enhancement. Moreover, heavy elements could contribute to higher X-ray attenuation coefficients. We investigated bismuth tetrathiotungstate nanoparticle which contains heavy metal bismuth and tungsten as a potential CT contrast agent. The results showed bismuth tetrathiotungstate nanoparitcle was non-toxic and has relatively high X-ray attenuation efficiency.

1.1 Materials

Gallium (III) nitrate hydrate (99.99%-Ga, Ga(NO3)3) was purchased from STREM

CHEMICALS with formula weight 255.73 g/mol. Potassium ferricyanide (99.8%,

K3[Fe(CN)6]) was purchased from Fisher Scientific with formula weight 329.25 g/mol.

Nitric acid （70% v/v, HNO3） was purchased from Sigma-Aldrich. Hydrochloric acid

(>37%, HCl) was purchased from Fluka. 2-Hydroxy-1,4-naphthoquinone (97%, C10H6O3) was purchased from Sigma-Aldrich. 1000 ppm calibration standards for AA were purchased from BUCK Scientific. Colon cancer cell line HT-29, Human bladder carcinoma cell line T24, and cervical cancer cell line Hela cells were purchased from

Sigma-Aldrich. The MTT Cell Proliferation Assay Kit containing two 15-mL MTT solution bottle was purchased from Trevigen.

1.1.1 carboxymethyl reduced polysaccharide (CMRD) preparation

The carboxymethyl reduced polysaccharide was produced through reaction of 25g dextran T10 with a reducing agent (0.4g of sodium borohydride) and 0.5 g of 50% (w/w) sodium hydroxide in 50 g of water at room temperature for 4 hours. Then 20 g 50% of sodium hydroxide and 6.95 g of bromoacetic acid was added to the solution and kept stirring for 16 hours at below 25oC. The temperature was controlled by iced bath. The product was washed by ethanol and ethanol/water mixture and collected by centrifugation.

1262 micromole carboxyl per gram was measured by titration.[30]

1.2 Methods

1.2.1 Atomic absorption spectroscopy (AA)

Atomic absorption spectroscopy is an analytical method for quantitative determination of metal elements using the absorption of optical radiation by free atoms in gaseous state. The sample is measured by atomic absorption spectrophotometer (Buck

Scientific 210VFP) under air/acetylene flow. Before sample measurement, 1000-ppm calibration standard solution was diluted serially to 5 different concentrations by using volumetric flask. Then a calibration curve could be drawn for calculating an unknown concentration based on sample absorption.

1.2.2 Acid base treatment method

Acid base treatment was used to decompose Ga[Fe(CN)6] bulk compound.

Ferricyanide anion was very stable in acidic condition, but it could be decomposed by strong base such as potassium hydroxide (KOH). 100 mg Ga[Fe(CN)6] bulk material was dissolve in ~ 2 mL 6 M KOH solution. After all the solid was dissolved under mild heating and cooled to room temperature, about 2 mL 8 M hydrochloric acid was added to neutralize the solution and to dissolve any oxide/hydroxide potentially formed. Then a small portion was taken out and diluted to measurable range for atomic absorption.

1.2.3 Cell Viability study

i) MTT assay.

MTT assay is a colorimetric assay using the MTT dye and plate reader to assess cell viability. The idea is that the enzyme NAD(P)H-dependent cellular oxidoreductase presents only in live cells has the ability to reduce the water-soluble tetrazolium dye

MTT, i.e. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan. Formazan is purple in color and soluble in DMSO. Color intensity can be measured by plate reader, so the cell viability can be calculated.

Protocol: Day1, 2×105 cells/ml were counted by using a haemocytometer and seeded in a 96-well plate. Each well has 100 uL McCoy’s medium (89%

McCoy’s+10%FBS+1% antibiotics) and incubated for 24 hours in a 37oC, 95%air and 5%

CO2 incubator. Day 2, nanoparticle stock solution was diluted with fetal bovine serum

(FBS) free McCoy’s medium. 100 uL of 5 different diluted NP solutions were introduced to 3 well/each. And 100 uL of free McCoy’s medium was added to all control cellular wells to maintain volume consistency. Day 3, after 24 hours of incubation, all nanoparticle/medium solutions were carefully removed by using a glass pipette and 100 uL of new McCoy’s medium was added to each well. 10 uL of yellow MTT dye solution was also added to each well and kept incubating for additional 3 hours. Then insoluble purple particle could be seemed after reduction. All MTT/media were carefully removed and 100 uL of DMSO was introduced to each well. After 5 min, all purple particles were

dissolved to give a purple solution. Finally, the 96-well plate was read by a plate reader

SpectraMaxM4 by Molecular Devices. And the intensity data were analyzed by Excel.

ii) Trypan Blue assay

Trypan blue ( C34H28N6O14S4) is a diazo dye which can selectively stain dead tissues or cells blue but it is ineffective to live cell. The idea is to count the number of live/dead cells in each plate, and calculate cell viability.

Protocol: Day 1, 100 uL of Hela cells in DMEM medium (89% DMEM+10%

FBS+1% antibiotics) were seeded in a 96 well plate and incubated for 24 hours in a 37oC

5% CO2 incubator. Day 2, NP solutions with 5 different concentrations were introduced to 3 well/each and incubated for another 24 hours. Day 3, all NP/DMEM medium was removed by using a glass pipette, and 100 uL of trypsin solution was added to each well to detach cells from the well. Re-suspending the cell and counting the live/dead cells were done by using a haemocytometer. The cell counting was repeated 2 times for each well. Based on the number of live vs. dead cells, the cell viability was calculated.

1.2.4 Dialysis

Dialysis was typically done using a regenerated cellulose tubular membrane with different pore sizes (MW3500, MW12000-15000) to purify the sample. The membrane has two openings and the opening could be either clamped by clamps or tied up by strings.

The sample was place inside of the membrane bag which was soaked in a large beaker

filled with distilled water. Free ions, small molecules, and uncoated polymers would pass through the pore and diffuses to the outside water. By changing the outside water, ionic by-products or unreacted materials could be washed off.

1.2.5 Elemental analysis

Samples were burned at 600 0C for 4 hours in a crucible in furnace to convert metals to their oxide forms. Then the metal oxides were dissolved in 70% nitric acid and diluted to their measurable range for atomic absorption spectroscopy.

1.2.6 Lyophilization

Lyophilization, also known as freeze-drying, is a dehydration process under vacuum. Frist, the sample solution was placed in a 600-mL freeze drying flask and frozen in actone/dry ice mixture. The drying was then carried out in duration of 8 to 12 hours using Labconco Freeze Dryer 8.

1.2.7 Kinetic study

Kinetic study was carried out to see how fast the sample compound could undergo ion-exchange with another metal ion. 500 mg Ga[Fe(CN)6] NPs were sealed in a dialysis bag (MW = 3500) and placed in a 100 mL solution containing 50 ppm FeCl2. During then ion-exchange process, 0.5 mL of sample solution was taken out at 0 min, 1min, 3 min, 5 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min, 120 min, 180 min, 240 min, 300 min. The sample solutions were diluted 10 times to 5 mL.

Meanwhile, 50-ppm FeCl2·4H2O solution was diluted to 1.5 ppm, 3.1 ppm, 6.25 ppm,

12.5 ppm, 25 ppm as the internal standard solutions for AA along with the commercially purchased 1000-ppm iron standard. Concentrations of samples were determined by AA, and the graph of ion-exchange with Fe2+ has been plot (figure 13). This procedure was repeated three times.

1.2.8 Thermal gravimetric analysis (TGA)

TGA is used as a thermal analysis method to measure weight loss of the sample as a function of increasing temperature. The thermal analysis was conducted using a TA instrument 2950 high-resolution thermogrvimetric analyzer (New Castle, DE, USA) in air for room temperature to 600 oC with a heating rate of 5 oC/min. A small amount of bulk sample was used for TGA and the weight loss was measured up to 600oC. Water molecule was lost first at around 200oC, and then the compound will decompose to metal oxide with the increasing temperature. So the number of water molecules can be calculated based of molecular formula.

1.2.9 Selectivity study

NP sample was soaked in a dialysis membrane bag and placed in 100 mL of a multi-metal solution at pH 1.86 made from magnesium nitrate hexahydrate

(Mg(NO3)2·6 H2O), zinc acetate dihydrate (Zn(OAc)2·2H2O), manganese (II) chloride tetrahydrate (MnCl2·4 H2O), Ferrous chloride tetrahydrate (FeCl2·4H2O), calcium chloride

(CaCl2) with each of the above ions at the ~ 100-ppm level for 24 hours. During this time,

these metal ions were competing with each other to exchange with gallium (III) to form more stable compound. Concentrations were determined and compared with the original multi metal solution to see concentration drop. Then the one exchanged most was consider 100% removal and the graph was re-plotted. This study was repeated three times.

1.2.10 Nanoparticle-surface conjugation of fluorescence dye molecules.

To prepare dye-labeled Ga NPs, 20 uL of 0.5 mM ethylenediamine solution was added to a 1 mL Ga NPs solution (about 1mM) under vigorous stirring overnight. The mixture was purified by dialysis to remove unbound ethylenediamine molecules. Next

1.15 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was added to 10 mL ethanol of carboxyfluorescene dye (0.5mM) solution and allowed to react for 24 hours.

Then ethylenediamine bound Ga NPs were reacted with 100 uL of dye/EDC solution for another 24 hours in dark. To remove the un-conjugated dye molecules, the product was dialyzed against distilled water for 2 days. Confocal fluorescence imaging of cells was performed with on an OLYPUS FV1000 IX8 confocal fluorescence microscopy to confirm the uptake of nanoparticles by cells.

1.2.11 X-ray attenuation measurements and CT phantom imaging

The linear X-ray attenuation coefficients of NP solutions at different concentrations, distilled water and air were measured using a Gamma Medica Xspect.

The CT phantom imaging studies were carried out with the following parameters: 512 slices/360o rotation; 75 kVp, 110 µA; field of view, 39.47, resolution, 150 micron. The

linear transformation of the raw data was performed to obtain the Hounsfield Units (HU) for the various concentrations of the NPs. For calibrations, the density value of deionized water is assigned as zero and used as the external reference. Four different concentrations of NPs dispersed in aqueous media and a sample of deionized water were used in the measurement performed with a Gamma Medica Xspect microCT scanner operating at 75 kVp and 110 µA.

1.2.12 Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FT-IR) analysis is a powerful technique for identification of the function groups based on vibration frequency. Burker Vector 33

Fourier Transform Infrared Spectrophotometer was used to characterizing the powder of products.

1.2.13 Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX)

TEM images provided information on morphology and the size of the NPs. One drop of acetone washed NPs solution was placed onto a carbon-coated TEM grid (400- mesh) which then allowed to air-dry prior to analysis. TEM measurements were made using a FEI Tecnai F20 transmission electron microscopy equipped with a field emission gun and analyzed at accelerated voltage of 200 kV and an emission current of 30 mA.

The EDX results were obtained for a selected area of the sample with the integrated scanning TEM unit and attached EDX spectrometer. EDX spectra showed all elements present in NPs.

Chapter 2: Novel Gallium Ferricyanide Nanoparticles as Potential Anticancer Drug

2.1 Abstract

Gallium ferricyanide nanoparticles were prepared using soluble gallium(III) ions and potassium ferricyanide. The carboxymethyl reduced dextran coated gallium ferricyanide nanoparticles are highly water dispersible, exhibit anticancer properties and can penetrate the cell plasma membrane to selectively remove iron (II) ions in the presence of other biologically essential metal ions including Mg(II), Ca(II), Mn(II),

Cu(II), and Zn(II) ions, suggesting that such NPs may have potential as a cell-permeable anticancer drug.

2.2 Introduction

Iron (Fe) is by mass the most common element on Earth and also abundant in biology. Iron is a required cofactor for many iron-related proteins and enzymes including hemoglobin, myoglobin, ferritin, catalase, lipoxygenases, that plays an important role in a variety of cellular processes like metabolism, respiration, and DNA synthesis [1,2]. It is indispensable mineral for human and pervasive in human food source. However, iron is a

redox active metal element. At physiological conditions, free ferric iron (Fe 3+) is able to produce free radicals and easily hydrolyzed to form insoluble hydroxide, both of which are generating toxicity to the cells. Thus, ferric iron has to be protein bonded and highly regulated in the body [3]. Ferric iron is reduced to ferrous iron (Fe2+) in intestinal lumen then dietary iron can be absorbed by the intestine [4]. The cell uptakes iron through ion channel or most efficiently through the so-called transferrin receptor-mediated endocytosis. Fe2+ can be oxidized to Fe3+ which would be chelated with transferrin protein. Then Fe3+ bounded transferrin protein is accepted by transferrin receptor and cell starts to engulf transferrin protein. This endocytosis process is mediated by the molecule clathrin. Clathrin is a large protein that assists in the formation of a clathrin coated pit

(~100 nm) on the inner surface of the plasma membrane of the cell. Then the clathrin coated vesicle is formed and enters cytoplasm of the cell, and clathrin will fall off from plasma membrane of the vesicle and return to cell membrane for recycle. The vesicle in cytoplasm is called early endosome. Once the early endosome is formed, proton (H +) is pumping into endosome through proton pump to lower the pH from 7.2 to 5.5. At such acidic condition, Fe 3+ is unbounded from transferrin protein and reduced to Fe 2+ by other protein molecules. Fe2+ can leave endosome through ion channel, but Fe3+ cannot. Then cellular Fe2+ is captured by a carrier protein for further cellular processing (Figure 1) [5].

Gallium (Ga) is a non-toxic and redox inactive metal. It is widely used in technological applications in modern society [6]. On the other hand, gallium has a long history of uses in the field of diagnostic and therapeutic medicine [7-9]. After the discovery of soluble platinum (IV) anticancer drug, the antineoplastic properties of

gallium were recognized in 1971, especially for treating bladder cancer and lymphoma

[10,19]. Late on, some gallium compounds have shown to be therapeutically efficient in treating several diseases and disorders other than just cancer. For example, gallium nitrate may be used to treat Paget’s disease of bone which is caused by abnormal bone remodeling [11]. Gallium (III) (Ga 3+) may be viewed as a mimic of ferric ion due to the similarities in chemical behavior with Fe3+, such as similar ionic radii (0.65 Å vs. 0.62 Å), electronegativity, ionization potential and electron affinity. Thus, Ga3+ can follow the Fe3+ route in vivo. It will occupy the Fe3+ centers in proteins and biomolecules. For example, it binds to transferrin protein and gets delivered by the similar iron uptake pathway (Figure

2) [12-14]. Therefore, water soluble gallium compounds can enter the cell and interferes with cellular iron metabolism [15-17]. For example, gallium ions bind to and inhibit the functioning of ribonucleotide reductase that is an enzyme important for DNA replication.

Non-functional ribonucleotide reductase would results in cell apoptosis and cell death

[18]. Gallium nitrate (Ga(NO3)3) and gallium chloride (GaCl3) were first reported as oral administrable gallium compounds for their anticancer properties [20]. On the other hand, gallium nitrate is an ionic compound which has low bioavailability. There are three possible ways that gallium could enter the cell: passive diffusion, pinocytosis and transferrin-mediated endocytosis. Passive diffusion is based on concentration gradient, which requires a very high extracellular concentration. Pinocytosis is a model of endocytosis and refers to as ‘cell drinking’, which is used for absorption of extracellular fluid. Unlike receptor-mediated endocytosis, pinocytosis is a nonspecific substance transport process that is randomly taken surrounding fluid contained ions. This process

also requires a high concentration in the extracellular fluid and is inefficient for cellular gallium ion uptake. Since ionic metal cations are easily cleared by kidney in a short amount of time, pinocytosis and passive diffusion are non-eligible for gallium uptake.

Only transferrin receptor-mediated endocytosis seems to be an option for gallium delivery. However, upon dissolution of gallium salts in water, the hard Lewis acid gallium cation is reapidly hydrolyzing to form the highly insoluble hydroxides. Large doses of gallium salts are known to generate renal toxicity due to such hydrolysis [21].

Moreover, treating cancer using soluble ionic gallium compounds requires too high gallium concentrations. Many researchers are investing organic complexes of gallium species to deliver gallium ions into cells in the hope that these compounds may be more effect and lower gallium concentrations may be required for cancer treatment.

Unfortunately, some cancer cells are starting to develop resistance for gallium [19]. There are two possible pathways for gallium resistance. For extracellular gallium ions, cancer cells could down regulate transferrin receptor activity to limit gallium ion intake. For intracellular gallium ions, some protein molecules in the cytoplasm could capture gallium ions and gallium ions would get pumped out through an ion pump on plasma membrane of cancer cells. So scientists are looking for new approaches to deliver gallium into cells for cancer treatment.

Gallium ferricyanide is an inorganic compound we have investigated for cancer treatment. This is idea came from the notion that there is an insoluble form of non-toxic

(III) (II) Prussian blue (Fe 4[Fe (CN)6]3 ) which was discovered not by a scientist but by a paint

maker Diesbach in Berlin around 1706 [22]. For a long period of time, Prussian blues was used as a paint pigment. The insoluble form of Prussian blue is an acid stable compound that can be simply made by mixing ferric ions (Fe3+) with ferrocyanide ions

(II) 4- ([Fe (CN)6] ). In the former times, people tended to believe that Turnbull’s blue

(II) (III) 2+ (III) 3- (Fe 3[Fe (CN)6]2) also could be made by reacting Fe with [Fe (CN)6] . And

Turnbull’s blue was thought to be stereochemically different from Prussian blue. Late on, by means of X-ray diffraction and electron diffraction studies, scientists found that the structure of Prussian blue and Turnbull’s blue were identical [23][24]. This means that

Fe(II) is always carbon-bound (i.e. often written as in the inner core of the formula) and

Fe(III) is always nitrogen-bound (i.e. often written as in the outside of the formula). The compound with a formula suggested by the Turnbull’s blue could not be made or detected.

One possible explanation is that Turnbull’s blue may be formed, but immediately undergoes inner electron transfer to convert itself into Prussian blue. From this analysis, we speculated that if the Fe3+ was replaced by its mimic ion Ga3+ in the synthesis of

Turnbull’s blue, electron transfer between Ga3+ and Fe3+ will not occurs. As the result, a

(III) 3- gallium analog of Turnbull’s blue may be formed. Moreover, the ligand [Fe (CN)6] in the gallium analog of Turnbull’s blue has the ability to give up Ga3+ for Fe2+ in order to from the more stable compound Prussian blue. This will probably help to capture Fe2+ ions inside the cell or on the cell membrane with the concomitant release of Ga3+ ions into the cell. Since iron is required for cell growth and especially for cancer cells, our ion- exchange strategy may be a unique method for delivering gallium into cancer cells, while

depleting iron source inside the cell at the same time to result in a better treatment of cancer.

Therefore, my goal was to design gallium ferricyanide nanoparticles as antitumor agent. Gallium ferricyanide was an insoluble compound that could be made as nanoparticles by polymer coating. In general, nanoparticles are more efficient and safer compared to ionic compounds containing the same metal ion at similar concentrations.

This is due to the so-called enhanced permeability and retention effect (the EPR effect).

The EPR effect is a phenomenon that nano-size particles tend to accumulate in tumor tissues much more than in normal tissues [25-27]. Because tumor cells grow very fast by which they have to stimulate the production of blood vessels to ensure nutrition and oxygen supply for growth. However, different from the regular newly formed blood vessels, these newly formed tumor vessels often grow in rush and thus are abnormal in architecture. The effective endothelial cells are poorly aligned with large pore or leaky walls which are ranging from 100 to several hundred nanometers. In contrast, normal vessel junctions are ranging from 5 to 10 nm [28]. (Figure 3). The size of proper nanoparticles designed for drug delivery usually is between 10 nm to 100 nm. Thus, nanoparticles enter cancer cells more efficiently and selectively. Except the transferrin receptor pathway, gallium ferricyanide nanoparticles could now be taken by endocytosis.

Fe2+ in cytoplasm would react with ferricyanide ligand to displace the gallium ion. Free gallium ions are released into cytoplasm to interfere with the cellular iron metabolism.

Fe3+ is released from transferrin protein and reduced back to Fe2+. Fe2+ can cross the membrane through ion channel and become available for other cellular process.

Figure 3. (up) Junction of endothelial cells in normal and healthy blood vessels is 5-10 nm. NP (>10 nm) would pass through. (Bottom) Junction of endothelial cells in abnormal blood vessels is usually few hundred nanometers, NP(<100 nm) would leak out from abnormal blood vessels and target tumor tissue.

2.3 Synthesis and characterization of bulk gallium ferricyanide

2.3.1 Method A

Ga(NO3)3 (aq) + K3[Fe(CN)6] (aq)  Ga[Fe(CN)6] (aq) + 3KNO3 (aq)

Bulk gallium ferricyanide (Ga[Fe(CN)6]) was synthesized by mixing 25 mM 40 mL gallium nitrate (0.2560g) solution with 25 mM 40 mL potassium ferricyanide

(0.3307g) solution under vigorous stirring for ~20 hours. A slightly cloudy yellow solution was obtained. Then the solution was transferred to a freeze-dry flask with the snap-on rubber cap and lyophilization was performed. After two days, all the water has been removed from the flask and a yellow-greenish powder was obtained. The impure powder was then sealed in a dialysis membrane bag (pore size MW = 3500) with both ends clamped. Then the dialysis bag was placed in 600 mL deionized water and the outside water was changed every 30 min and 8 times. During this time conductivity of the outside water was measured until it reached the level of deionize water. A small amount of wet sample was taken out and dried on watch glass for TGA determination. Drying of the compound was achieved by lyophilization. Sample A was characterized by IR and atomic absorption.

1 Ga[Fe(CN)6] bulk A

0.9

0.8

0.7 Ga[Fe(CN)6 ] bulk

0.6 Transmittance, Transmittance, %

0.5

0.4

4,000 3,001 2,002 1,003 -1 Wavenumber, cm

Figure 4. Fourier transform infrared spectroscopy (FT-IR) of the Ga[Fe(CN)6] bulk compound synthesized by method A.

Figure 5. TGA of Ga[Fe(CN)6] bulk compound synthesized by method A. Weight lost below 200 ºC showed on the first drop on graph was due to the water lost, The second drop showed on graph above 200 ºC may due to cyanide lost. 21% weight loss was due to the water.

2.3.2 Method B

Ga(NO3)3 (aq) + H3[Fe(CN)6] (aq)  Ga[Fe(CN)6] (aq) + 3HNO3 (aq)

10 mL of 3.3025 g K3[Fe(CN)6] was soaked in about 10 g of

Amberlite® IR120 Resin (hydrogen form) for 10 min and filtered. The filtrate was collected and soaked in another 10 g resin for 10min. This progress was repeated three times to insure all potassium ions were exchanged with hydrogen ions. To examine all potassium ions were removed, the AA analysis was performed to detect potassium ions in solution.

In order to get gallium ferricyanide bulk compound, 40 mM 50 mL gallium nitrate solution was added to 40 mM 50 mL H3[Fe(CN)6] solution (pH was measured to be 1.02).

At low pH, hydrogen cyanide gas may be formed and released. Ethylenediamine was added to neutralize the acidic solution to pH 3.41 and the solution began to become cloudy. The solution was stirred in 2 days and light yellow cloudy solution was obtained.

The solution was transferred to a freeze-dry flask and lyophilized was performed under vacuum for 2 days. The dried powder was transferred into a dialysis membrane bag with both ends lamped. The sealed dialysis membrane bag was placed in 600 mL deionized water and the water was changed every 2 hours for 3 days until no colored solution was leaked out. A small portion was took out and dried on a watch glass for TGA analysis.

The rest of sample B was lyophilized again to remove water. The dried bulk compound B was characterized by atomic absorption and IR.

Figure 6. TGA of Ga[Fe(CN)6] bulk compound synthesized by method B. Weight lost below 200 ºC showed on the first drop in the graph was due to the water lost. The second drop showed in the graph above 200 ºC may be due to cyanide lost. 21%weight loss was due to the water.

Ga[Fe(CN)6] bulk B 1

0.9

0.8

0.7

0.6 Transmittance,% 0.5

0.4 4,000 3,001 2,002 1,003 Wavenumber, cm -1

Figure 7. FT-IR of Ga[Fe(CN)6] bulk compound synthesized by method B.

Figure 8. The Fe(III) center donates electron through a d orbital to empty π*, an antibonding orbital of the CN ligand.

Figure 9. The formula used for calculating water molecules in crystal lattice. X represents the number of water molecules.

2.3.3 Results and discussion of Ga[Fe(CN)6] bulk compound

Elementary analysis was used to determine iron to gallium ratio in the bulk compound synthesized by method A and method B. However, there was some residual solid leftover that could not be fully dissolved in 70% nitric acid after the sample was decomposed at 600 ºC for 4 hours. One possibility was that not all gallium and iron was transferred to oxide, and carbon bound iron may form acid stable carbide at such a high temperature. So acid-base treatment method was used to determine metal ratio in the formula.

In the bulk compound A, a large amount of potassium ions were detected by atomic absorption. So the formula of bulk compound A could be written as KxGa1-x/3

[Fe(CN)6]. This could be explained by using a crystal lattice model. Gallium, which was

+3 charged, was sitting in the octahedral holes and leaving all the tetrahedral holes empty.

In order to minimize the void space in the tetrahedral sites, there is a big chance for the

+1 charged potassium ions to fill some of some of the tetrahedral holes in crystal lattice as found in the so-called soluble Prussian blue structure. Thus, potassium could be incorporated in compound A as a counter ion, not as a contaminant.

To exclude potassium ions, method B was developed. All potassium ions were removed by ion-exchange with resin, and atomic absorption was used to ensure that no potassium ions are found in the H3[Fe(CN)6] solution. Then compound B was treated by acid base treatment method and analyzed by atomic absorption. The iron to gallium ratio was 1:1.2 which was closing to the expected 1:1 ratio. So the formula for bulk compound

B could be written as Ga[Fe(CN)6].

Free cyanide (CN) vibration frequency in sodium cyanide (NaCN) was at 2178 cm-1[29]. However, once iron(III) binds to carbon in the cyanide ligand, electrons were back donated from the d orbitals in iron(III) to π*, the antibonding orbital of the CN ligand (Figure 8). The CN triple bond is thus weakened, which lows the CN stretching vibration frequency. Bulk compound A may contained some impurities as seen in its IR spectrum, in addition to the CN vibration frequency at 2116 cm-1. Several peaks at the low frequency range could not be explained (Figure 4). Bulk compound B shows a clean

IR spectrum which contains the same CN vibration frequency at 2116 cm-1 (Figure 7).

There was also a shoulder at 2206 cm-1 may due to atmosphere carbon dioxide C=O vibration.

TGA for both bulk compound A and B showed 21% water loss from crystal lattice (Figures 5 and 6). The number of water molecules in the crystal lattice could be

calculated (Figure 9). Based on these calculations, 4.1 moles of water molecules are present in each mole of Ga[Fe(CN)6]. So the modified molecule formula for the bulk compound B should be Ga[Fe(CN)6]·4H2O.

2.4 Synthesis and characterization of gallium ferricyanide nanoparticles

2.4.1 Synthesis of gallium ferricyanide nanoparticles

CMRD

Ga(NO3)3 (aq) + K3[Fe(CN)6] (aq)  Ga[Fe(CN)6] (aq) + 3KNO3 (aq)

Gallium Ferricyanide nanoparticles (Ga[Fe(CN)6] NPs) are synthesized by a one- step reaction. Nanoparticles could be formed by mixing 100 mL 1mM 50ul 70% nitric acid treated gallium nitrate (0.0257g) aqueous solution with 1.3522g CMRD contained

100 mL 1mM potassium ferricyanide (0.0337g) aqueous solution. The orange yellow mixture was stirred for 24 hours. Then volume of the mixture was reduced by lyophilization. And unreacted starting materials and by-products were purified by dialysis for 2 days. Finally, a greenish yellow powder was gathered by lyophilization. Gallium ferricyanide nanoparticles were characterized by transmission electron microscopy

(TEM), IR, ion-exchange kinetic studies, and metal selectivity studies. Additionally, the

NPs were examined for cellular toxicity by cell viability studies using the MTT assay and cell uptake was evaluated by confocal microscopic imaging studies.

2.4.2 Results and discussion

Different coating agents were investigated. These include polyvinylpyrrolidone

(PVP), polysaccharide (dextran), and carboxymethyl reduced dextran (CMRD). Only

CMRD could successfully stabilize gallium ferricyanide nanoparticles (Ga NPs).

Compared to dextran, CMRD has lower toxicity and higher water solubility [30]. It was an excellent polymer for coating nanoparticles for biomedical engineering studies. By tuning the synthetic method, Ga NPs could be made with great water solubility and stability. FT-IR was shown as in Figure 10 to confirm that CMRD was present and coated on Ga NP-surfaces. Due to a heavy coating, the peak intensity of CN group in Ga NPs were weak. The TEM images (Figure 11) showed that the average size of gallium ferricyanide nanoparticles were ~25 nm. Ion-exchange selectivity studies (Figure 12) suggested that the ferricyanide ion forms the most stable compound towards Fe2+ in the presence of other divalent ions. This is in disagreement with the Irving-Williams Series which refers to the relative stabilities of a complex formed by a metal ion. The Irving-

William series states that the stability constant for the formation of a complex follows the order: Mn(II)Zn(II) [31]. However, the violation could be

2+ 3- explained based on the following redox chemistry: once Fe reacts with Fe(CN)6 , the immediate electron transfer will take place to form Prussian blue, the most stable compound among all possible metal ferricyanide- or ferrocyanide compounds. This is probably the reason that the ferricyanide ligand has the highest selectivity towards ferrous ions. To investigate how fast Fe2+ could be removed by Ga NPs, kinetic study was performed (Figure13, top). In the first half hour, Fe2+ was removed significantly. And at

120 min, the solution reached saturation. When the data were processed, the iron removal reaction within the first 20 min can be fit into a pseudo-first order reaction (Figure13, bottom).

0.9

0.8

0.7 GaPB BulkB GaPB NPs 0.6

Transmittance,% CMRD 0.5

0.4 4,000 3,001 2,002 1,003 Wavenumber, cm -1

Figure 10. FT-IR of gallium ferricyanide nanoparticles in comparison with gallium ferricyinde bulk compound and the CMRD polymer coating agent

Figure 11. Transmission electron microscopy (TEM) images of CMRD coated gallium ferricyanide nanoparticles with an average size of 25 nanometers.

120 Selectivity study 100

Ionremoved, % 40

0 Fe Cu Zn Ca Mn Mg

Figure 12. Selectivity studies. Ga NPs undergo ion-exchange with 100 ppm of several

M(II) ions for 24 hours showing that Fe(II) to be the most selective metal and

Mn(II)/Mg(II) the least selective metals.

70 Kinetic study 60

40 ],ppm

2+ 2+ 30 [Fe

0 0 50 100 150 200 250 300 Time,min

1.8 1.6 1.4

1.2 1 0.8 y = -0.0247x + 1.6762 Ln[Fe2+] 0.6 R² = 0.9806 0.4 0.2 0 0 5 10 15 20 25 time,min

Figure 13. Kinetic study. (Top) Fe2+ removal by Ga NPs vs. time. (Bottom) Within first

20 min, the removal of Fe2+ fits the pseudo-first order reaction.

Confocal microscopy was used to investigate if Ga NPs could actually enter the cancer cell. Both transferrin receptor rich Human bladder carcinoma cell line T24 and most common human colon cancer cell line HT-29 were used as models in this study. A carboxylate fluorescence dye was attached to the Ga NP surfaces through the EDC coupling (figure 14A). If the NPs entered the cell, fluorescence signals would be detected by confocal microscopy while the dye itself could not enter the cell on its own and the excess Ga NPs could be washed away before the imaging process. The confocal images showed that the Ga NPs could penetrate cell cytoplasm within 3 hours (Figures 14B and

15). Cellular toxicity of the Ga NPs were examined by cell viability studies shown in

3+ Figure 16. The CD50 was measured to be about 3.2 mM [Ga ] of Ga NPs. Compared to the ionic compound Ga(NO3)3 , Ga NPs were more efficient. Figure 17 showed clear view of cell growth/killing at different Ga NPs concentrations.

Figure 14. A: preparation of fluorescence Ga NPs with EDC coupling. B: Confocal microscopy images of T24 cells: (Top left) fluorescence image of cells incubated with dye-conjugated Ga NPs for 3 hours; (Top right) bright field images of cells incubated with dye conjugated Ga NPs for 3 hours; (lower left).

140 Cell Viability study 120

100

60 Ga[Fe(CN)6] NPs Ga(NO3)3

40 Cell Viability, Cell Viability, % 20

0 1.58 2.36 3.15 3.94 4.73 5.51 6.30 -20 [Ga3+ ],mM

Figure 16. Cell viability curve based on the MTT assay. Effect of Ga NPs on viability of

HT-29 cells after 24 hour-incubation in comparison with the cell viability using Ga(NO3)3.

Figure 17. Bright field light microscopic images. Various concentrations of Ga NPs used for incubating HT-29 cells for 24 hours. A: 1.58 mM; B: 2.3 mM; C: 3.15 mM; D: 3.94 mM; E: 4.73 mM; F: 5.51 mM; G: 6.3 mM; H: NP-free control cells.

2.5 Conclusion

We have developed a stable and biocompatible nanoplatform for delivering gallium ions into cancer cells as a potential new treatment for cancer. Ga NPs are more efficient in cancer cell killing and have the ability to enter the cancer cells independently of the transferrin-receptor mediated pathway. Therefore, the novel gallium ferricyanide nanoparticles might be a better choice for developing gallium-based anticancer drugs.

Chapter 3: Gallium Lawsonate Nanoparticles as Potential Anticancer Agent

3.1 Abstract

Naphthoquinones could produce reactive oxygen species and generate oxidative stress in the hypoxic region of tumor. One of such naphthoquinones, 4 naphthoquinone

(lawsone) is a great metal chelator with potential anticancer activity. In this study, Ga- lawsonate nanoparticles were investigated for their anticancer properties.

3.2 Introduction

Gallium is the second metal after platinum has shown therapeutic activity in cancer treatment (see chapter 2). In order to prevent gallium ions from hydrolysis, to improve bioavailability, and to facilitate membrane penetration, many gallium complexes have been developed as potential anticancer drugs [21,32]. Currently, there are two gallium complexes, gallium maltolate and tris (8-quinolinolato) gallium (III) (KP46) that have been evaluated in clinical settings as oral administration drugs [21]. Gallium maltolate, or tris (3-hydroxy-2-methyl-4H-pyran-4-onato) gallium (III) (Figure 18) was designed as the analog of ferric maltol complex. Maltol is a naturally occurring organic

compound used to increase the oral bioavailability of iron [33] and gallium [32]. Gallium maltolate was found to be safe, tolerable and officiate as an oral anticancer drug in Phase

I clinical trial. Similarly, KP46 (Figure 19) was also evaluated to be officiate with high oral gallium bioavailability for use in cancer treatment. These two gallium coordination compounds have opened a new revenue for development of new gallium complexes with potential antitumor activities [34]. By looking at the structure of maltol, we hypothesized that gallium cation, as a hard Lewis acid, should form stable complexes with the hard soft

Lewis donor atoms of oxygen in other similar ligands. So we searched a suitable organic molecule in the naphthoquinone family that uses oxygen donor atoms to form a gallium complex in the same manner as found in gallium maltolate to investigate its anticancer activities.

Current therapy to treat solid tumor relies mainly on the use of chemotherapeutic agents, radiotherapy, or the combination of both. On the one hand, these two therapies are capable of killing tumor cells in well-oxygenated regions of tumor. On the other hand, these therapies have little or no effect on malignant stem cells of tumor in hypoxic areas

[35]. This is because drugs can be rapidly taken up by cells in aerobic regions than oxygen insufficient regions. Inadequate blood supplies to tumor tissues results in poor oxygen delivery, which is called hypoxia [36]. Due to hypoxia, chemotherapeutic drugs are poorly delivered to tumor tissues that are often hindered to achieve drug cytotoxic concentration levels [37]. Radiotherapy is also ineffective in hypoxic regions because of low oxygen tension inhabit production of highly reactive oxygen radicals necessary for cytotoxicity [38]. The alkylating agents such as mitomycin C (MC) have been found to

have preferential cytotoxicity to hypoxic malignant cells. MC belongs to the quinone family and requires bioreducive activation to produce reduction that can exploit the hypoxic environment [39].

Quinones are ubiquitous in nature and popular in clinical therapy as anticancer drugs, such as doxorubicin, anthracyslines, daunorubicin, mitoxantrones, mitomycin, and saintopin. Quinones express cytotoxicity towards cancer cells in various mechanisms including arylation, alkylation, intercalation, redox reaction, and generation of free radicals [40, 41]. 1,4-naphthoquinone and its derivatives are some important examples from the quinone family. These compounds are drawing more and more attention for their various uses. They are biodegradable and widely used in pharmaceutics, agrochemicals, and other functional chemicals. For example, the vitamin K family is a group of 1,4-naphhoquinone derivatives. Vitamin K family is known as potential blood clotters, antibacterial, antifungal, and anticancer agents [42]. Focus on its anticancer properties, the cytotoxic effects of 1,4-naphthoquinone and its derivatives are mainly coming from two aspects. Firstly, 1,4-naphthoquinone and its derivatives are able to inhibit DNA topoisomerase-II. DNA topoisomerase-II is an important enzyme that cuts both strands of DNA helix simultaneously to manage DNA tangles and supercoils.

Secondly, 1,4-naphthoquinone and its derivatives could produce oxidative stress by redox cycling and act as alkylating agents [43, 47]. This is a general property of naphthoquinones. Free radicals may be generated by either one or two electron reduction.

One electron reduction of a naphthoquinone produces semiquinone by enzymes including ubiquinone reductase (NADH), cytochrome P450 reductases (NADPH), and cytochrome

b5 reductase. Semiquinones are very unstable and could react with oxygen at the physiological pH to produce reactive oxygen species (ROS) which would cause damages to the cell [44]. Two electron reduction of naphthoquinones generates hydroquinones by enzyme quinone oxidoreductase (NAD(P)H) [45,46]. As a small organic molecule, 1,4- naphthoquinone may not be able to penetrate the cell membrane to be taken up efficiently by cells. Furthermore, the bioavailability of 1,4-naphthoquinone as a drug may also limited because of its poor water solubility. Therefore, many potential anticancer drugs containing quinone moieties have been converted to metal complexes that are then dissolved in DMSO for in vitro cell testing [48].

Lawsone (2-hydroxy-1,4-napthoquinone) (Figure20) is a natural compound originally found in henna plant leaves as a red-orange dye. It is a monohydroxy naphthoquinone and poorly soluble in water. It is an effective chelator of divalent or trivalent metal ions because of its juxtaposed phenolic hydroxyl and keto groups [49,35].

Gallium is a trivalent metal with anticancer activities. Lawsone is a great ligand to chelate and deliver gallium to tumor tissues. Meanwhile, lawsone could be activated at hypoxia tumor cells. Novel gallium lawsonate nanoparticles (Ga-lawsonate NPs) were therefore investigated for their anticancer properties.

Figure 18. Structures of the oral gallium maltolate (right) and maltol.

Figure 19. Structure of the oral KP46.

Figure 20. Structure of lawsone.

3.3 Synthesis

Lawsone (0.0522g) was dissolve in 20 mL of 90% ethanol. 0.0207g of potassium carbonate was added to deprotonate phenolic alcohol group of lawsone to generate potassium lawsonate. The solution was left in air to dry. After ethanol was evaporated, 20 mL of deionized water was added to completely dissolve potassium lawsonate. Next, 100 mL 1 mM gallium nitrate (0.0255g) was added to potassium lawsonate solution under vigorous stirring (Figure 21). Brown precipitate was formed after addition. The mixture was reacted for 1 hour and filtered by vacuum filtration. The Ga-lawsone solid was washed with 10 mL of deionized water 3 times. The solid was left to dry in vacuum for

10 min and then dried in air overnight. The product was collected as dry powder. 100 mg of Ga-lawsonate powder was weighted out and dissolved in about 1 mL of DMSO in a

1.5 mL Eppendorf centrifuge tube under sonication. Meanwhile, 2 g of PVP 8000 was

dissolved in 10 mL of deionized water. 50 uL of Ga-lawsonate DMSO solution was added into the previously prepared 10 mL PVP solution under vigorous stirring for 30 min. The NPs were purified by dialysis. There was some NPs leak out from membrane

(MW3500) and most of them remained that indicates gallium lawsonate was formed as nano-platform. If it was small molecules, all gallium lawsonate particles would leak out from dialysis bag. The Ga-lawsonate NPs were treated with concentrated nitric acid and the concentration of gallium was determined by AA. Cytotoxicity of Ga-lawsonate NPs on human colon cancer HT-29 and Human urinary bladder carcinoma cell line T24was determined by the MTT method(Figure 22). Potassium lawsonate was also tested on T24 cell line for comparison (Figure 23).

Figure 21. Synthetic scheme and proposed structure of gallium lawsonate.

120 Ga-lawsonate HT29

100

40 Cell Viability, Cell Viability, %

0 15 29 44 58 73 88 [Ga 3+], um

80 Ga-lawsonate T24 70

50 40

30 Cell Cell Viability,% 20 10 0 800 700 600 500 400 300 200 100 [Ga3+],uM

Figure 22. Cell viability studies of Ga-lawsonate NPs on HT-29 Cells (Top) and T24 cells (Bottom) by MTT method.

K-lawsonate

100

20 Cell Cell viability,% 10

0 1.3 1.66 2 2.3 2.6 2.86

[K+],mM

Figure 23. Cell viability studies of Potassium lawsonate on T24 Cells.

3.4 Results and discussion

Ga-lawsonate NPs were treated with concentrated nitric acid. Gallium ion was shown to coordinate by lawsone using AA analysis. Furthermore, we have developed a simple bi-phasic method to prepare Ga-lawsonate NPs. Crystal growth was tried using the hydrothermal method at different temperatures, solvents, and molar ratios. For example, hydrothermal reactions at different temperatures such as 80 °C, 90 °C, 100 °C,

110 °C, 120 °C, and 140 °C were all tested. We found that lawsone would decompose at higher temperatures (>100C). Different solvents such as water, water/ethanol, methanol, and methanol/water were used in these hydro(solvo)thermal reactions without success.

Diffusion method using DMSO/ether was also tried in several reactions that lasted 3 months. Thus far, we have been unable to produce diffraction-quality single crystals for

X-ray structure determination of this compound. Gallium is a trivalent metal ion and lawsonate is a bidentate ligand. So the proposed structure of Ga-lawsonate should have octahedral geometry. The cytotoxicity of Ga-lawsonate NPs on HT-29 cells and T24 cells were different. The CD50 of Ga-lawsonate NPs were about 60 uM on HT-29 cells and 300 uM on T24 cells. Compared with potassium lawsonate (CD50=2.3mM ) , Ga-lawsonate were more efficient in cancer cell killing. Our preliminary studies show that Ga- lawsonate NPs are much more cytotoxic than the Ga NPs, suggesting a possible synergistic effect. However, more detailed studies should be performed on the Ga- lawsonate project to confirm such synergy before this compound can be developed as an oral anticancer drug.

3.5 Conclusion

In summary, Ga-lawsonate NPs could be readily synthesized to investigate the potential synergy in anticancer activities. Furthermore, the known bioreductive properties of lawsone in the Ga-lawsonate NPs could be exploited to target hypoxic tumor cells to enhance the effectiveness of the drug for treating more malignant tumors. The future work on this project should include in vivo animal testing of this compound as an oral drug, biodistribution of the NPs and biological half-life of such NPs.

Chapter 4. Bismuth Tetrathiotungstate Nanoparticles as Potential Contrast Agent

for Computed Tomography

4.1 Abstract

Novel ultrasmall Bi2(WS4)3 NPs with PVP-coating were developed as a potential cellular CT contrast agent. Cytotoxicity, cellular uptake and potential use of the PVP- coated ultrasmall Bi2(WS4)3 NPs as a CT contrast agent were investigated. Owing to the combined X-ray attenuation effect of bismuth and tungsten, the NPs exhibit a very high

CT value based on the bismuth molarity as compared to other nanoparticle-based CT contrast agents.

4.2 Introduction

Computed tomography (CT) is a powerful noninvasive technique for diagnosing diseases, disorders and biological functions by producing 2D and 3D cross sectional images of the human body using X-ray radiations. Due to the variation of X-ray attenuation, contrast can be readily generated between different areas such as soft tissues and organs. CT contrast agents are usually used as “dyes” to highlight specific areas.

Various CT contrast agents have been found and many of them are iodinated organic compounds [50,51]. One of the characteristics of CT contrast agent is high X-ray

attenuation coefficient which contributes to visualization enhancement. In addition, heavy elements can contribute to higher X-ray attenuation coefficients. Inorganic compounds with heavy metals should have higher X-ray attenuation coefficients than current iodinated CT contrast agents. Several compounds with metallic elements of high atomic numbers including silver [52, 53], cesium [54], bismuth [55, 56], thorium [57-60], and tungsten [61] were investigated for potential contrast applications due to their high

X-ray attenuation power. Only few of them have succeeded to become clinical contrast agents. For example, almost 100 years ago, barium sulfate (BaSO4) was first introduced as aqueous suspensions for imaging gastrointestinal (GI) tract [62]. It has used in clinical with a little or no change in the formulation for a long time [63]. On the other hand, thorium (Z=90) is the second heaviest naturally-occurring element after uranium (Z=92).

A suspension of ThO2 showed superb image quality with virtually no acute toxicity or any immediate side-effects [58, 59]. Hence, an X-ray contrast agent (Thorotrast®, ThO2 ) was introduced in 1928 and quickly developed in applications for imaging cerebral arteries, liver, spleen, lymph nodes, and other organs [57-60]. However, the potential toxicity would later result in tragic consequences [64]. Thorium has a naturally-occurring radioactive isotope that is an alpha-emitter with an extremely long radioactive half-life

10 (4.08 MeV, t1/2= 1.41×10 yr). Unusually long biological half-life of the ThO2 colloidal formulations (t1/2= ca.22 yr) would later cause liver cancer and leukemia in millions of patients who were injected with Thorotrast® in Europe, North America and Japan

[65,66].

Bismuth (Bi, Z=83) compounds are usually low in toxicity or nontoxic, nonradioactive, and biocompatible. Tungsten (W, Z=74) compounds are also low in toxicity or nontoxic, nonradioactive, and biocompatible. Therefore, the tetrathiotungstate

2- (WS4 ) anion can be used as a biocompatible ligand to form an insoluble 3D coordination polymer with Bi(III). In this way, metal ions are impossible to dissociate from the polymer network, which makes it safe after the compound is delivered in vivo as

2- a CT contrast agent. The idea of developing a novel CT contrast agent using MS4 (M =

Mo and W) as a ligand came from commercially available copper removing drug ammonium tetrathiomolybdate ((NH4)2MoS4) which is used to treat Wilson’s disease.[67]

Molybdenum and tungsten are from the same group. As such, they share similar properties and can form isostructural compounds. Consequently, we chose the tetrathiotungstate ligand to investigate a novel potential CT contrast agent. Bismuth tetrathiotungstate has been synthesized as particles in the nanosize region, and then coated with biocompatible polyvinylpyrrolidone (PVP) in order to have superior water solubility. The solution stability, cytotoxicity, cellular uptake and X-ray attenuation properties are investigated.

4.3 Synthesis

3(NH4)2WS4(FM) + 2Bi(NO3)3(FM/aq,PVP)  Bi2(WS4)3 NPs + 6NH4NO3

First, 0.0522g ammonium tetrathiotungstate (NH4)2WS4 was dissolved in 10 mL of formamide (FM). 20ml containing 0.2 g of PVP8000 and 4 g of sodium citrate were added to 20 mL of bismuth nitrate Bi(NO3)3 (0.0485g) formamide solution. Then

(NH4)2WS4 solution was added into Bi(NO3)3 solution dropwise under vigorous stirring for 4 hours at room temperature. The mixture was purified by dialysis in distilled water and dark brown/black Bi2(WS4)3 NP powder was gathered after lyophilization. Bi2(WS4)3

NP powder has great water dispersability that could be easily used in characterization and cell testing.

4.4 Results and Discussion

Bi2(WS4)3 NPs would form in cube shape and the energy dispersive X-ray spectrum (Figure 24, bottom) showed all three elements (Bi, W, S) are present in the NPs.

The size of Bi2(WS4)3 NPs ranges from 2 to 10 nm (Figure 24,top). Confocal images showed that the NPs can penetrate cells (Figure 25). And cell viability studies by the

Trypan blue method showed that Bi2(WS4)3 NPs are nontoxic at micro molar concentration level (Figure 26). Phantom images (Figure 27) showed that with increasing Bi2(WS4)3 NPs concentration images get brighter. This means that Bi2(WS4)3

NPs could produce a positive contrast. The X-ray attenuation of PVP-coated Bi2(WS4)3

NPs were measured and reported as density values in Hounsfield units (HU). As shown in Figure 28, the CT values expressed in HU exhibit a linear relationship with the Bi(III) concentration in NPs. The slope of this linear curve indicates the X-ray attenuation efficiency of the contrast agent in the unit of HU/mM. In comparison, the PEGylated gold

NPs gave the value of 5.3 HU/mM [68] while this value for PVP-coated Bi2S3 was reported to be 9.3 HU/mM [69]. Moreover, the CT value was measured and reported for a polymer-coated TaOx NP system to be 6.0 HU/mM [70]. It is clear that our PVP-coated

Bi2(WS4)3 NPs appear to have the highest value among all these nanoparticle systems (10

HU/mM) based on the Bi(III) molarity because of the combined X-ray attenuation effect of bismuth and tungsten.

Figure 24. (TOP) Transmission electron microscopy (TEM) image of Bi2(WS4)3 NPs on the 5-nm scale. (Bottom) Energy dispersive X-ray spectrum of Bi2(WS4)3 NPs; TEM analysis revealed that the nanoparticles are well-formed cubes and the size distribution appeared to be relatively wide, ranging from 2 to 10 nm.

Figure 25: Fluorescence microscopic images of HeLa cells incubated with dye-labeled

NPs for 3 hours: (right) Bright-field image, (left) confocal fluorescence microscopy image; Carboxyfluorescene dye was conjugated to the surfaces of the ethylenediamine- coated Bi2(WS4)3 NPs by the EDC-coupling reaction. Fluorescent signal in the cytoplasm of cells confirms the uptake of the NPs by the cells. Therefore these agents can serve as intracellular contrast agents.

Figure 26. Histogram showing the viability of Hela cells in the presence of various amounts of Bi2(WS4)3 NPs after 24 hours of incubation as determined by the Trypan Blue exclusion method.

Figure 27. Phantom images of Bi2(WS4)3 NPs at different concentrations.

Figure 28: The CT values vs. Bi(III) concentrations.

4.5 Conclusion

In summary, the use of bismuth and tungsten in a single compound could raise X- ray attenuation power while the combination surpasses most of the known inorganic nanoparticulate systems so far reported in the literature for CT contrast applications. The main goal of the current studies is to develop novel nanoparticulate CT contrast agents.

Bi2(WS4)3 NPs were nontoxic and have high CT values, suggesting that such NPs may have potential for cellular CT imaging and image-guided drug delivery applications.

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